Small molecule enzyme inhibitors serve as essential drugs for the treatment of a wide range of pervasive and life threatening diseases. The starting compounds needed for the development of these inhibitors are most often either identified by high throughput screening (HTS) or are provided by natural substrates. However, for several classes of enzymes such as proteases and phosphatases neither approach is very effective. A powerful new method for small molecule inhibitor discovery called Substrate Activity Screening (SAS) has enormous potential to accelerate the development of enzyme inhibitors. The SAS method, which is the first substrate-based method for fragment discovery and optimization, consists of three steps: (1) a diverse library of low molecular weight substrates is screened against the enzyme target to identify hit fragments, (2) the identified substrate fragments are optimized, and (3) the optimized substrates are converted to inhibitors by direct incorporation of inhibitor pharmacophores. Screening for substrate as opposed to ligand fragments has two key advantages. First, false positives often seen in traditional high throughput inhibitor screens are eliminated because the assay requires productive substrate binding and turnover. Secondly, catalytic substrate turnover results in signal amplification enabling the identification of weakly active lead fragments. The overall objective of this application is to develop and apply the SAS method to three important enzyme classes - cysteine proteases, phosphatases and arginine deiminases. Our central hypothesis is that the SAS method can be used for the reliable and efficient discovery of small molecule inhibitors that will be exceptionally useful for chemical biology, pharmacology and drug discovery applications. A number of highly significant advances and positive impacts will result from inhibitors that have already been developed or are proposed, including: (A) Advancement of cathespin S imaging agents for clinical use in ex vivo tumor imaging for real time identification of tumor margins during surgery. (B) Advancement of novel, potent and orally available inhibitors of the protease cruzain that already have been shown to eliminate symptoms of acute Chagas' disease in animal models. (C) Advancement of caspase inhibitors to Huntington's disease animal models that have already been shown to rescue neurons from cell death in Huntington's disease ex vivo models and that cross the blood-brain barrier. (D) Advancement of potent and selective inhibitors of the phosphatase STEP to cell and animal models of Alzheimer's disease. (E) Advancement of small molecule inhibitors that will help to define the biological roles of protein arginine deiminases, which catalyze a post translational modification implicated in diseases such as rheumatoid arthritis. Many innovations will result from the implementation and application of the SAS method to the aforementioned enzymes. The expected outcomes will be robust and efficient strategies for the development of exceedingly useful small molecule inhibitors. Considerable positive impacts are also anticipated given the enormous biological and therapeutic importance of the specific enzymes being targeted.